Project Summary/Abstract. The clinical signature of botulinum neurotoxin (BoNT) is peripheral neuromuscular blockade and flaccid paralysis, which depending on the serotype (A-G) can last for months. BoNTs are the most toxic proteins known to man and have been classified by the Centers for Disease Control and Prevention as one of the six highest-risk biothreat agents. Despite high potential as a bioterrorist weapon, BoNTs are widely used in medical and cosmetic procedures (i.e., Botox). However, even under a controlled environment unwanted side effects have been reported and in some cases severe life-threatening disorders. Clinical intervention of BoNT poisoning is complicated by not only its extreme toxicity but also its long half-life (months for BoNT/A). Currently a botulinum heptavalent antitoxin (BAT) is the only approved medical intervention, yet, has limited value since antibodies can only neutralize circulating toxin, which is negated once cellular poisoning takes place. There are no pharmacologic antagonists available that act once cellular intoxication takes place and none that even advanced to Phase I Clinical Trials. Prohibitive as developing a therapeutic might seem we have shown that BoNT/A intoxication can be attenuated using a synergistic combination of an antibody and a pharmacological antagonist. As significant as this finding has been to the BoNT field, a further mastery would be the use of singular pharmacological agents fashioned to attenuate BoNT/A's toxicity. We currently possess several classes of small molecule inhibitors that can intercede at three junctions critical to BoNT's pathology: SNARE protein cleavage, neurotoxin endocytosis, and blockade of acetylcholine release. From this arsenal we have discovered molecules engendered to block multiple processes associated with BoNT/A neurotoxicity. Impressively, this dual inhibitor-mechanism strategy provides the first small molecule that can extend time to death from a BoNT/A post exposure occurrence. We have established how potassium channel blockade can provide full sustenance in the reversal of paralysis for post- intoxication of BoNT/A. For this research we offer the molecules 3,4-diaminopyridine (3,4-DAP) and 3,4,5- triaminopyridine (3,4,5-TAP); both are highly effective for BoNT/A rescue. However, aminopyridine's effectiveness is severely compromised by their short half-life. We will address this limitation within our proposed studies. Finally, as a means to augment our small molecule pharmacological antagonist research initiative we will use medicinal chemistry, X-ray crystallography and pharmacokinetics to develop both greater selectivity and more potent inhibitors against the botulinum neurotoxins. The successful integration of our research goals will bring the complex and challenging problem of treating botulinum toxicity toward a clinically viable treatment.